阅读说明：本技术 一种鱼胶原蛋白的高效制备方法 (Efficient preparation method of fish collagen ) 是由 安信生 裘耀东 唐治国 于 2019-11-22 设计创作，主要内容包括：本发明涉及一种鱼胶原蛋白的制备方法,包括如下步骤：步骤S1、鱼鳞前处理,步骤S2、微波辅助超声提取,步骤S3、提纯、后处理。本发明采用双频超声、双频微波辅助提取相结合的制备方法,这种方法结合了现有技术中各种鱼胶原蛋白的制备方法的优点,鱼胶原蛋白的产率更高,纯度更好,制备成本更低廉,过程中使用的原料来源广泛,制备工艺简单易操作,适合规模化生产,制备得到的胶原蛋白产品质量高,产率大；在提取前,采用不同性质的脱脂清洗液进行清洗,清洗效果更好,有利于脂的溶解,对后期提取有促进作用,使得制备得到的鱼胶原蛋白纯度更高。(The invention relates to a preparation method of fish collagen, which comprises the following steps: step S1, fish scale pretreatment, step S2, microwave-assisted ultrasonic extraction, and step S3, purification and post-treatment. The invention adopts a preparation method combining double-frequency ultrasound and double-frequency microwave-assisted extraction, the method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw material used in the process has wide sources, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning liquids with different properties are adopted for cleaning, the cleaning effect is better, the dissolution of fat is facilitated, the later-stage extraction is promoted, and the prepared fish collagen is higher in purity.)

1. The efficient preparation method of the fish collagen is characterized by comprising the following steps:

step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to obtain a precipitate;

step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;

step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.

2. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution in step S1 is 1 (0.2-0.4): 0.1-0.3): 10-20): 8-10): 10-12.

3. The efficient preparation method of fish collagen according to claim 1, wherein the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-.

4. The efficient preparation method of fish collagen according to claim 1, wherein the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.

5. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the fish scales to the extracting solution in step S2 is 1 (20-30).

6. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in step S2 is 1 (20-30) to (10-15).

7. The method for efficiently producing fish collagen according to claim 1, wherein the extract is at least one of water, ethanol, and ethyl acetate.

8. The method for efficiently preparing fish collagen according to claim 1, wherein the decolorant is at least one selected from the group consisting of activated carbon, fly ash and chitosan.

Technical Field

The invention relates to the technical field of biological products, in particular to an efficient preparation method of fish collagen.

Background

Collagen is a natural protein synthesized by fibroblasts in an animal body, is a structural protein of extracellular matrix, is also called collagen, and widely exists in connective tissues such as bones, skins, tendons and the like of animals, so that tissues and organs have good mechanical structures, thereby playing roles in supporting the organs and protecting the organism. The collagen and its degradation products are rich in glycine, serine, alanine, aspartic acid and other natural moisturizing factors and a large amount of hydrophilic groups, so that it has a moisturizing function. In addition, collagen has good permeability, can be filled between skin matrixes and absorbed by the skin, and has the effects of repairing the skin and whitening the skin, so that collagen and collagen peptide which is a degradation product of the collagen are more and more widely applied to beauty care and cosmetics.

The fish collagen is collagen produced by taking fish as a raw material, so that the quality of the collagen is improved, the source of the collagen raw material is enlarged, waste is changed into valuable, and resources are saved. The existing methods for producing fish collagen mainly comprise a hot water extraction method, a salt method, an acid method, an alkaline method, an enzyme method and the like. The reaction condition for producing collagen by the enzyme method is mild, the triple helix structure of the collagen is not damaged, and the method can keep the characteristics and is widely applied. However, the existing enzymolysis technology is carried out at normal temperature and normal pressure or even at high temperature and high pressure, the enzymolysis, concentration and drying processes all need higher temperature, the influence on the activity of the fish collagen is larger, the product quality is difficult to ensure, the powder yield is lower, and the small-batch production cannot be realized. In addition, the existing production methods also have the defects of higher production cost, poor quality of finished products, longer production period of products, larger volume of extracting solution, incomplete impurity removal of enzymatic hydrolysate and larger enzyme dosage.

Therefore, how to efficiently prepare high-quality collagen is a technical problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a high-efficiency preparation method of fish collagen for overcoming the defects of the prior art; the preparation method adopts a preparation method combining double-frequency ultrasound and double-frequency microwave-assisted extraction, combines the advantages of various fish collagen preparation methods in the prior art, has higher yield and purity of fish collagen, lower preparation cost, wide raw material sources used in the process, simple and easy operation of the preparation process, suitability for large-scale production, and high quality and yield of the prepared collagen product; before extraction, degreasing cleaning liquids with different properties are adopted for cleaning, the cleaning effect is better, the dissolution of fat is facilitated, the later-stage extraction is promoted, and the prepared fish collagen is higher in purity.

In order to achieve the purpose, the invention adopts the technical scheme that:

step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to take precipitates;

step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;

step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.

Further, in step S1, the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution is 1 (0.2-0.4): (0.1-0.3): 10-20): 8-10): 10-12.

Further, the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-.

Further, the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.

Further, in the step S2, the mass ratio of the fish scales to the extracting solution is 1 (20-30).

Further, the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1 (20-30) to 10-15.

Preferably, the extracting solution is at least one of water, ethanol and ethyl acetate.

Further, the decolorizing agent is selected from at least one of activated carbon, fly ash and chitosan.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the preparation method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw materials used in the process are wide in source, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning solutions with different properties are adopted for cleaning, the cleaning effect is better, lipid dissolution is facilitated, and the later extraction is promoted, so that the prepared fish collagen has higher purity, the preparation process is safe, sanitary and harmless, and the social value and the economic value are higher.

Detailed Description

The invention relates to step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to take precipitates;

step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;

step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.

Further, in the step S1, the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution is 1 (0.2-0.4): 0.1-0.3): 10-20): 8-10): 10-12; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.

Further, in the step S2, the mass ratio of the fish scales to the extracting solution is 1 (20-30); in the step S2, the mass ratio of the crude extract to the sodium chloride solution to the glacial acetic acid is 1 (20-30) to 10-15; the extracting solution is at least one of water, ethanol and ethyl acetate; the decolorizing agent is selected from at least one of activated carbon, fly ash and chitosan.

The preparation method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw materials used in the process are wide in source, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning solutions with different properties are adopted for cleaning, the cleaning effect is better, lipid dissolution is facilitated, and the later extraction is promoted, so that the prepared fish collagen has higher purity, the preparation process is safe, sanitary and harmless, and the social value and the economic value are higher.

The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto: